The aim of this project is to understand more fully the process of DNA replication through detailed study of the mechanisms of selected DNA polymerases and factors which affect these enzymes. Experiments will concentrate on measurement of basic parameters of enzyme mechanism in vitro, specifically including processivity, i.e., the number of nucleotides added each time the polymerase binds DNA. We have recently developed a quantitative assay for measurement of processivity of DNA synthesis in vitro (Bambara et al., (1978), J. Biol. Chem. 253, 413.) The availability of this assay provides an opportunity to examine factors which perturb DNA polymerases, specifically DNA replication-associated proteins. It is the major goal of this project to use such kinetic assays to detect and isolate DNA replication associated proteins in cell and viral systems where genetic tools are not available for this purpose. We will therefore begin a search of calf thymus cell extracts for proteins that increase the extent of processive DNA synthesis by purified calf alpha DNA polymerase. We will also search by the same means for stimulatory proteins related to the avian myeloblastosis virus (AMV) DNA polymerase.